Aberrant overactivation of WNT signaling appears to play a major role in Idiopathic Pulmonary Fibrosis (IPF). Inhibitors of overactivated WNT signaling are candidates for therapy of IPF. USP34, a novel de-ubiquitylating enzyme, has been identified as an IPF target in the WNT/(-catenin pathway. It interacts with Axin and is required to de-ubiquitylate Axin, allowing it to go to the nucleus where it increases (-catenin signaling. Cellular validation experiments have demonstrated that USP34 regulates WNT positively via nuclear localization of (-catenin. As WNT signaling is elevated in IPF, a USP34 inhibitor would decrease signaling and have a potentially therapeutic effect. Progenra has developed a novel isopeptidase assay platform for measuring DUB activity, and the work proposed here will utilize this assay to discover inhibitors of USP34 by high throughput screening. First, the assay will be configured for USP34 in a 384 well plate format. Next, it will be employed to screen three small molecule libraries - the ChemDiv Protease Library (2000 compounds), the Maybridge HitFinder diversity library (14,000 compounds), and the DiverSet Library (20,000 compounds) - for potent and selective inhibitors of USP34. The efficacy of selected hits will be evaluated in a fibrosis cell-based model of WNT signaling and in cytotoxicity and cellular proof of concept assays. In later stages, lead compounds will be evaluated in in vivo models (bleomycin treatment of mice) and pharmacokinetics studies. The commercial goal is a pharmaceutical agent active against IPF. PUBLIC HEALTH RELEVANCE: The WNT/(-catenin signaling pathway has been linked to various human diseases, including cancer and Idiopathic Pulmonary Fibrosis (IPF). For these diseases, inhibitors of WNT signaling are potentially useful as therapeutic agents. Progenra proposes to collaborate with academic experts in the WNT pathway and lung fibrosis to discover inhibitors of an enzyme called USP34, which acts to increase WNT signaling in IPF. Inhibitors of USP34 will be identified in high throughput screening using Progenra's assay technology and evaluated for preclinical development as therapeutic agents to treat IPF.